The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. A project, done in collaboration with Dr. Fumihito Ono, resulted in the development of a zebrafish neuronal system suitable for investigating sodium and calcium channel modulation and function during a development stage that leverages the genetic malleability and optical transparency of this model organism. Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available. We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven green fluorescent protein were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch- clamp studies. Two kinetically distinct tetrodotoxin-sensitive sodium currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating sodium currents were preferentially expressed in relatively large neurons, while slowly-inactivating sodium currents was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab, and zscn5Laa are possible candidates for these sodium current components. Voltage-gated calcium currents were primarily comprised of a high-voltage activated component arising from omega-conotoxin GVIA-sensitive CaV2.2 (N-type) calcium channels. A few neurons displayed a minor low-voltage-activated component. Calcium currents in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability. Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies. Won YJ, Ono F, Ikeda SR. Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons. PLoS One. 2012;7(8):e42602. A second study examined an endogenous ligand for the G-protein coupled receptor GPR18. Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analog of anandamide, is an endogenous ligand of GPR18. However, other studies failed to detect activation of GPR18 by NAGly. To address this inconsistency, we investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. We heterologously expressed GPR18 in rat sympathetic neurons and examined the modulation of N-type calcium channels. Proper expression and trafficking of receptor was confirmed by the rim-like fluorescence of fluorescently-tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents, but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported by our laboratory (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors, G protein-coupled inwardly-rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple pathway utilizing to Gs, Gz, or G15. These results argue that NAGly is not an agonist for GPR18 or that GPR18 signaling involves non-canonical pathways not examined in these studies. Lu, VB, Puhl, HL, Ikeda, SR. N-arachidonyl glycine (NAGly does not activate G protein-coupled receptor 18 (GPR18) signaling via canonical pathways. Manuscript in revision. A third project, currently nearing first phase completion involves the investigation of a recently de-orphanized G-protein coupled receptor termed GPR41 or FFAR3 in rodent sympathetic neurons. These receptors use short-chained fatty acid (SCFA; e.g., acetate and propionate) as endogenous ligands but little else is known. We have discovered that GPR41 is natively expressed and functionally coupled to calcium channels in sympathetic neurons (primarily the celiac/superior mesenteric ganglia). We have also generated evidence that receptor expression is highest (and possibly functional confined to) paravertebral and prevertebral sympathetic neurons. These findings are potentially important for several reasons. First, GPR41 maybe expressed in neurons that innervate adipose tissue and thus be an important target for anti-obesity drugs. Second, acetate is a major metabolite of ethanol. Hence, GPR41 may be involved in both the response to ethanol (especially hangover symptoms) and addiction. It is possible that polymorphisms in GPR41 contribute to susceptibility to alcoholism in humans. Third, as GPR41 is likely capable of detecting ethanol indirectly via blood acetate levels, it may be possible to leverage this property to interrupt neural pathways contributing to addiction in model organisms using genetic techniques.